proquestdocuments 2015-07-26

aftar isi
1. Effects of Systemic Fluoride and in vitro Fluoride Treatment on Enamel Crystals....................................... 1
26 July 2015 ii ProQuest

Effects of Systemic Fluoride and in vitro Fluoride Treatment on Enamel Crystals
Link dokumen ProQuest
Abstrak Systemically administered fluoride at a concentration of 75 ppm increases the surface roughness of
developing enamel crystals in rats, which may be significant in advancing our understanding of the biological
mechanism of fluorosis. Thus, the aim of this study was to investigate whether the increased surface roughness
may be a result of surface restructuring by the direct action of fluoride at the crystal surface. We examined the
fluoride dose-dependent roughening of enamel crystal surfaces in vivo, in the rat, and whether this roughening
could be mimicked by the in vitro treatment of rat enamel crystals with neutral pH fluoride solutions. Our results
showed that enamel crystal surface roughness increased after treatment with increasing fluoride ion
concentrations, whether applied in vitro or administered systemically. This suggests a mechanism, alongside
others, for the increased surface roughness of crystals in fluorotic enamel.
Teks lengkap Headnote
ABSTRACT
Systemically administered flouride at a concentration of 75 ppm increases the surface roughness of developing
enamel crystals in rats, which may be significant in advancing our understanding of the biological mechanism of
fluorosis. Thus, the aim of this study was to invesligate whether the increased surface roughness may be a
result of surface restructuring by the direct action of fluoride at the crystal surface. We examined the fluoride
dose-dependent roughening of enamel crystal surfaces in vivo, in the rat, and whether this roughening could be
mimicked by the in vitro treatment of rat enamel crystals with neutral pH fluoride solutions. Our results showed
that enamel crystal surface roughness increased after treatment with increasing fluoride ion concentrations,
whether applied in vitro or administered systemically. This suggests a mechanism, alongside others, for the
increased surface roughness of crystals in fluorotic enamel.
KEY WORDS: dental fluorosis, enamel crystals, fluoride, AFM.
INTRODUCTION
With the introduction of fluoride as the main anticaries agent used in preventive dentistry, and perhaps an
increase in fluoride in our food chain, dental fluorosis has become an increasing world-wide problem (Aoba and
Fejerskov, 2002). There is still debate regarding the precise mechanisms causing dental and skeletal fluorosis
(Bawden et al., 1995; Aoba and Fejerskov, 2002; Robinson et al., 2004a,b).
Recently, increases in the roughness of crystal surfaces in developing fluorosed enamel have been reported,
and the implications of this with regard to fluorosis discussed (Kirkham et al., 2001; Robinson et al., 2003,
2004b). The proposed mechanism of how these roughened crystals may cause fluorosis involved impairment of
crystal growth due to changes in crystal surface chemistry, and/or incomplete removal of modulating matrix
proteins, which are known to hinder crystal development (Aoba and Fejerskov, 2002; Robinson et al., 2004b).
Rougher crystals have been shown to bind increased amounts of protein in other tissues (Gathercole et al.,
1996).
Reasons for the increased roughness were thought to be a change in kink and step-site density, reflecting a
higher supersaturation level in tissue fluid for the deposition of mineral. This might arise due to the deposition of
less soluble fluoridated mineral species-apatite. Retention of matrix proteins themselves, due to increased
interaction with the altered crystal surfaces, might also generate altered growth patterns. Increased retention of
matrix protein is highly likely, since proteins both bind more effectively to fluoridated apatite in vitro (Bawden et
al., 1995; Aoba and Fejerskov, 2002) and are retained in naturally fluorotic enamel to a significant degree
(Tanabe el al, 1988: DenBesten, 1999; Robinson et al., 2004b).
Recent studies in our laboratory have showed, with atomic force microscopy (AFM), that ameloblastin adsorbs
26 July 2015 Page 1 of 8 ProQuest
to crystal surfaces (Misch et al., unpublished data), and that dentin phosphoprotein is bound more tightly to
fluorotic enamel crystals than to non-fluorotic ones (Spencer et al., unpublished data). Thus, since fluorotic
enamel crystals appear to have a greater binding capacity for the matrix proteins, which would lead to their
impaired removal, this would offer an explanation for the greater concentration of proteins and impaired crystal
growth in fluorotic enamel, which, in turn, results in the production of hypomineralized enamel.
It is well-established that fluoride administration in vivo generates rougher crystal surfaces, and we
hypothesized that this particular effect could be due, at least in part, to interaction between crystal surfaces and
fluoride in the surrounding tissue fluid. In view of this, we carried out in vivo studies to establish whether the
degree of roughening of the surfaces was dependent on the amount of fluoride given. The in vitro studies were
carried out to demonstrate that the crystal surface restructuring (roughness) could be caused by fluoride and,
therefore, could be a mechanism, alongside others, in the development of the roughened crystal surface in
fluorosed enamel. The crystal surfaces were visualized by AKM, which is capable of imaging features at the
size of the apatite unit cell (Kirkham et al., 1998, 2001).
MATERIALS &METHODS
Small particles (approximately 50-100 μg in weight) of maluration-stage enamel were microdissected free from
the underlying dentin and used for the isolation of individual crystals. These particles were obtained from Wistar
rats which had received 25, 50, or 75 ppm of fluoride (added as NaF) to the drinking water on day 22, and
continuing for 21 days. The presence of fluorotic enamel was determined by the appearance, after eruption, of
striae on the enamel. Matched littermates were the controls, which received nonfluoridated drinking water for
the same period of time, and were fed the same laboratory diet. (The animal use protocol was reviewed and
approved by the University's committee on Use and Care of Animals.)
Isolation of the Enamel Crystals
All detectable traces of matrix protein were removed from the enamel samples by a sequential extraction
procedure described previously (Kirkham et al., 1998). The enamel crystals were sonicated for 2 min in HPLC-
grade methanol to reduce aggregation. Approximately 5 μL of this suspension was then pipetted onto freshly
cleaved mica. The methanol was removed by evaporation, leaving a coating of dispersed hydroxyapatite
crystals on the surface. The specimens were dried in a desiccator for at least 12 hrs and subsequently
examined by AFM.
Protein-free developing enamel crystals were obtained from the maturation stage of normal rat incisors. These

enamel crystals were treated with: (a) 0 ppm, 25 ppm. 50 ppm, and 75 ppm NaF solutions [pH 7.4, phosphate-
buffered saline (PBS), containing 0.01% NaN^sub 3^) in a water bath held at 37°C for 21 days; or (b) 0, 200
ppm, 1000 ppm, 2000 ppm, 10,000 ppm, and 20,000 ppm NaF solutions (pH 7.4, PBS buffer plus NaN^sub 3^)
in a water bath held at 37°C for 18 hrs. The volume of NaF solution relative to the weight of enamel crystals was
approximately 1.5 mL solution/0.1 mg enamel crystals. After centrifugation, the crystals were washed 6 times
with de-ionized water (pH 7.4) and dispersed onto a mica surface prior to surface roughness measurements
with a Nanoscope IIIa AFM.
Atomic Force Microscopy
All samples were imaged in tapping mode in air, with a Nanoscope IIIa Multimode AFM and controller (Digital
Instruments, Santa Barbara, CA, USA) equipped with a 120 x 120 μm; J-scanner. Commercially available
tapping mode cantilevers, TESP, were used (Digilat Instruments. Santa Harbara. CA, USA) (thickness, 2.6-4.5
μm; width, 28-30 μm; height, 10-15 μm; length, 125 μm; resonant frequency, 297-378 kHz; spring constant, 24-
61 N/m; nominal tip radius of curvature, 5-10 nm). Multiple images for each sample were obtained with scan
sizes ranging from 500 x 500 nm to 10 x 10) μm at 70-80% of free cantilever amplitude. The phase image
results from effective dumping or alteration to the resonant frequency of the cantilever, depending on the
modulus of the surface being examined. Therefore, it is more sensitive to the surface morphology change and
gives much higher resolution.
RESULTS
To reduce the number of AFM images, we chose: maturation control (Fig. 1a); 50 ppm administered
systemically (Fig. 1b); and 50 ppm (Fig. 1c) and 1000 ppm (Fig. 1d) applied in vitro as being representative of
the crystal surface morphology.
Fluoride Administered in vivo
When phase images were viewed, crystal surface nanoscale changes became more evident as the
concentration of fluoride increased (Fig. 1b). In crystals from rats receiving systemic fluoride for 21 days, this
resulted in a significant change in surface roughness of the maturation crystals (Fig. 2), from R^sub a^ = 0.53
±0.18 in the control to 0.65 ±0.21 (25 ppm F), 0.71 ±0.20 (50 ppm F), and 0.85 ±0.28 (75 ppm F). The R^sub a^
of the crystals from the rats receiving the 25 ppm and 50 ppm fluoride systemically were not significantly

different from each other. Crystals from rats receiving 75 ppm were significantly rougher (P <0.05) than crystals
from those receiving 25 ppm or 50 ppm.
Fluoride Administered in vitro
Fig. 1c shows the height and phase images of maturation-stage enamel crystals after in vitro treatment with 50
ppm F, pH 7.4, for 21 days at 37°C. There was no significant difference in the roughness of the crystals that
were in vitro-treated with 25 ppm, 50 ppm, or 75 ppm fluoride (R^sub a^, 0.46 ±0.16, 0.46 ±0.17, and 0.45
±0.17, respectively) for 21 days and the non-treated control (R^sub a^ = 0.46 ±0.17) (Fig. 2). Treating the
enamel crystals with higher concentrations of NaF (200 ppm, 1000 ppm, 2000 ppm, 10,000 ppm, 20,000 ppm
fluoride for 18 hrs at 37°C) produced considerable visual nanoscale changes to crystal surface morphology (Fig.
1d). [A time-course experiment in which enamel crystals were treated in vitro with NaF at these higer
concentrations showed that surface roughening occurred after 18 hrs (data not shown).] Surface roughness
measurements confirmed this observation, with R^sub a^ 0.45 ±0.17 (control) and 0.62 ±0.20 (200 and 1000
ppm F) increasing progressively to 0.75 ±0.21 (2000 ppm F), 0.93 ±0.29 (10,000 ppm F), and 1.01 ±0.31
(20,000 ppm F) (Fig. 2).
DISCUSSION
Our results showed that fluoride, administered both systemically to rats and used as an in vitro treatment of rat
enamel crystals, was able to change the surface structure of the crystals, resulting in increased surface
roughness. When fluoride was administered systemically at 75 ppm, the maturation-stage enamel was
significantly rougher than control maturation-stage enamel crystals, in agreement with Kirkham et al. (2001).
However, we also showed that the roughness of maturation-stage enamel crystal surfaces increased at much
lower fluoride concentrations (25 ppm), and, although, there was no significant difference in roughness between
the 25-ppm and 50-ppm treatments, there was a further significant increase in roughness when 75 ppm was
administered. This observation suggested a dose-dependence of crystal surface roughness by systemically

administered fluoride, although this did not appear to be linear at the dosages used.
There is evidence that, during the systemic administration of fluoride, concentrations during the transition and
maturation stage of enamel development may be very high (Weatherell et al. 1977: DenBesten and Thariani,
1992), and that this fluoride may be labile (Weatherell et al., 1977). In view of this, we also investigated the
direct effect of fluoride on enamel crystal surfaces in vitro. Enamel crystals in vitro were exposed to F
concentrations of 25 to 75 ppm for 21 days. Unlike systemic administration, this did not cause any significant
increase in the surface roughness of the crystals.
At higher concentrations in vitro (> 200 ppm), however, increases in crystal surface roughness did occur. This
raises the possibility that effective F ion activity in vivo may be very high in the immediate crystal environment.
This, however, assumes that the roughness originates in precisely the same way both in vivo and in vitro.
It has been suggested that increased roughness in vivo was due to fluoride incorporation into depositing
mineral, possibly flouride substituting for hydroxyl ions in apatite, producing a less soluble phase (Robinson et
al., 2004a,b). The effective increase in supersaturation could lead to increased density of kink and step growth
sites on the crystal surface. However, analysis of the in vitro data suggests another possibility.
Since the in vitro treatments were not carried out in the presence of solutions supersaturated with respect to
apatite, surface roughness could not originate directly from growth-related phenomena. It might be that some
restructuring of the crystal surface occurs, perhaps via ions in the Gouy-Chapman layers and those in the
crystal surface. This process would include ion exchange, dissolution, and/or redeposition. While the precise
chemistry is not known, this would result from fluoride altering the levels of supersaturation in the supernatant,
with respect to dissolving and reprecipitating phases of the crystal surface. For example, incorporation of
fluoride into the crystals by hetero-ionic exchange would effectively increase supersaturalion with respect to the
surface. This may in turn reduce surface carbonate. Calcium would also be released to maintain charge
balance, resulting in reprecipitation of less soluble phases. There is also evidence that these crystals may be
heterogeneous from a chemical viewpoint, which would give rise to growth on some areas but not in others
(Robinson et al., 2004a). At high concentrations of fluoride in vitro, double decomposition could occur between
fluoride and hydroxyapatite. resulting in calcium fluoride and various calcium fluoride complexes (Øgaard,
2001). Whether this precise mechanism occurs in vivo is difficult to say, since solution conditions in vivo are not
precisely known. However, such changes cannot he ruled out, especially at the maturation stage of
development, where fluoride is known to accumulate. Therefore, in vivo, these phenomena would occur if
concentrations in the crystal eruironment reached such high levels.
On the basis of the known pharmacokinectics of fluoride (DenBesten and Crenshaw, 1984; Aoba and
Fejerskov. 2002; Robinson et al., 2004b), it seems unlikely that, at 25 to 75 200 ppm fluoride given systemically
would generate greater than 200 ppm free fluoride in the enamel organ or enamel matrix. However, it is
possible that the concentration of fluoride at the enamel crystal surface during growth could be very high.
DenBesten and Crenshaw (1984) reported that the maturation stage of fluorosed enamel from rats that had
received 75 ppm systemically contained approximately 405 ±99 ppm of fluoride. These concentrations were
obtained from analyses of whole enamel and are not indicative of even higher concentrations that might occur
at the enamel crystal surface.
The precise concentrations of fluoride in supernatant solutions in vivo are unknown, particularly at this
developmental stage, where it is known that fluoride concentrates. In addition, in the immediate vicinity of
crystal surfaces, concentrations of all ions, including fluoride, would be considerably higher than in the solution,
depending upon the surface zeta potential. We therefore suggest that if fluoride concentrations of >200 ppm are
reached at the enamel crystal surface after the systemic administration of fluoride, they could cause crystal
surface changes similar to those seen after in vitro application. Crystal roughening in vivo may therefore be
partly due to supersaturation effects on crystal growth, and partly a direct effect on surface restructuring.
In conclusion, this study has showed that both in vitro and systemically administered fluorides are able to

restructure the surface of the enamel crystal. The topography of the crystal surfaces was altered by treatment
with fluoride. The enamel crystal surface roughness increased after treatment with increasing fluoride ion
concentrations.
ACKNOWLEDGMENTS
This investigation was supported by USPHS Research Grant DE015599 from the National Institute of Dental
and Craniolacial Research, National Institutes of Health, Bethesda, MD 20892.
Sidebar
Received September 7, 2005; Last revision March 29, 2006; Accepted July 18, 2006
References
REFERENCES
Aoba T, Fejerskov O (2002). Dental fluorosis: chemistry and biology.
Crit Rev Oral Biol Med 13:155-170.
Bawden JW. Crenshaw MA. Wright JT, LeGeros RZ (1995).
Consideration of possible biologic mechanisms of fluorosis. J Dent Res 74:1349-1352.
DenBesten PK (1999). Biological mechanisms of dental flourosis relevant to the use of fluoride supplements.
Community Dent Oral Epidemiol 27:41-47.
DenBesten PK. Crenshaw MA (1984). The effects of chronic high fluoride levels on forming enamel in the rat.
Arch Oral Biol 29:675-679.
DenBeslen PK, Thuriani H ( 1992). Biological mechanisms of fluorosis and level and timing of systemic
exposure to fluoride with respect to fluorosis. J Dent Res 71:1238-1243.
Gathercole LJ. Swan AJ. Price G. Dieppe PA (1996). Nanometre-scale surface features of arthropathic
microcryslals and their relation to protein adsorption: a study by scanning probe microscopy and wide angle x-
ray diffraction. J Mater.Sci Mater Med 7:511-516.
Kirk ham J. Brookes SJ. Shore RC. Bonass WA. Smith DA. Wallwork ML. et al. (1998). Atomic force microscopy
studies of crystal surface topology during enamel development. Connect Tissue Res 38:91-100.
Kirkham J. Brookes SJ, Zhang J. Wood SR, Shore RC. Smith DA, et al. (2001). Effect of experimental fluorosis
on the surface topography of developing enamel crystals, Caries, Res. 35:50-56.
Øgaard B (2001). CaF(2) formation: cariostalic properties and factors of enhancing the effect. Caries Res
35(Suppl 1 ):40-44.
Robinson C. Brookes SJ, Wood SR, Kirkham J. Shore RC (2003). The effect of fluoride on the developing
mineralised tissues: a brief review. Oralpropphylaxe 25:33-38.
Robinson C. Connell S. Kirkham J, Shore RC, Smith A (2004b). The effect of flouride on the developing tooth.
Caries Res 38:268-276
Tenabe T, Aoba T, Morena EC, Fukae M (1988) Effect of flouride in the apatitic lattice on adsorption of enamel
protiens onto calcium apatites. J Dent Res 67:536-542.
Wallwork ML, Kirkham J. Chen H. Chang SX. Robinson C, Smith DA. et al. (2002). Binding of denlin
noncollagenous matrix proteins to biological mineral crystals: an atomic force microscopy study. Calcif Tissue
Int 71:249-255.
Weathcrell JA. Deutsch D, Robinson C, Hallsworth AS (1977). Assimilation of fluoride by enamel throughout the
life of the tooth. Caries Res 11(Supp1 1):85-115.
AuthorAffiliation
H. Chen1, A. Czajka-Jakubowska2, N.J. Spencer1, J.F. Mansfield3, C. Robinson4, and B.H. Clarkson1*
1 Department of Cariology, Restorative Sciences and Endodontics, University of Michigan School of Dentistry,
1011 N. University, Ann Arbor, MI 48109-1078, USA; 2 Karol Marcinkowski University of Medical Sciences,
Dept. of Conservative Dentistry and Periodontology, Poznan, Poland; 3 University of Michigan Electron
Microbeam Analysis Laboratory; and 4 Leeds Dental Institute, Division of Oral Biology, Leeds England; *

MeSH Administration, Oral, Animals, Cariostatic Agents -- administration & dosage, Crystallization, Microscopy,
Atomic Force, Rats, Rats, Wistar, Sodium Fluoride -- administration & dosage, Surface Properties -- drug
effects, Cariostatic Agents -- adverse effects (utama), Dental Enamel -- drug effects (utama), Fluorides, Topical
-- adverse effects (utama), Fluorosis, Dental -- etiology (utama), Sodium Fluoride -- adverse effects (utama)
Substansi Cariostatic Agents; Fluorides, Topical; Sodium Fluoride;
Judul Effects of Systemic Fluoride and in vitro Fluoride Treatment on Enamel Crystals
Pengarang Chen, H; Czajka-Jakubowska, A; Spencer, N J; Mansfield, J F; et al
Judul publikasi Journal of Dental Research
Volume 85
Edisi 11
Halaman 1042-5
Tempat publikasi Houston
Hak cipta Copyright American Association for Dental Research/American Academy of Implant Dentistry Nov
2006
_______________________________________________________________
Hubungi ProQuest

proquestdocuments 2015-07-26

Documents